The invention relates to a method for the cold sterilization of preparations containing blood coagulation factor VIII.
Congenital or acquired blood coagulation disorders attributable to a deficiency or insufficiency of plasmatic coagulation factors can be treated with preparations containing these factors in a concentrated form. These concentrates include fractions prepared from blood plasma. The fractions most commonly used at this time are Cohn fraction I obtained by alcohol fractionation according to Cohn (J. Am. Chem. Soc., vol. 72 (1950), p. 465), a cryoprecipitate obtained by cold precipitation, a fraction containing factor VIII (antihemophilic globulin=AHG), and an AHG concentrate containing antihemophilic globulin and the prothrombin complex, i.e., factors II, VII, IX and X (PPSB). Will all of these preparations there exists the risk of transmitting hepatitis.
The radioimmune assay for hepatitis B surface antigen (HBsAg) is 100 to 1000 times too insensitive, so that a high percentage even of HBsAg-negative blood products, especially AHG and PPSB preparations is infectious and capable of transmitting hepatitis B (cf. J. H. Hoofnagle et al., "The prevalence of Hepatitis B Surface Antigen in Commercially Prepared Plasma Products", J. Lab. Clin. Med., vol. 88 (1976), pp. 102 to 112, and R. J. Wyke et al., "Transmission of Non A-Non B Hepatitis to Chimpanzees by Factor IX Concentrates after Fatal Complications in Patients with Chronic Liver Disease", The Lancet 1 (1979), no. 8115, pp. 520 to 524).
Since it is not possible by diagnostic measures to obtain hepatitis-safe blood products from plasma pools, the sterilization of blood and blood components acquires central importance. Human albumin, for example, is made hepatitis-safe by pasteurization, i.e., by heating for 10 hours at 60.degree. C. Coagulation factors, however, cannot be pasteurized, because they are destroyed even by one hour of heating at 60.degree. C. Therefore, a method of sterilization would have to be developed which would operate at low temperatures rather than high. Such a method was proposed by LoGrippo (cf. G. A. LoGrippo et al., "Chemical Sterilization of Whole Blood and Plasma with Beta-Propiolactone", Hepatitis Frontiers, Henry Ford Hospital, Int. Symposium 1956, Little, Brown and Co., Boston, Mass. (1957), pp. 371 to 385, and G. A. LoGrippo et al., "Chemical and Combined Methods for Plasma Sterilization", Bibl. Haematol., vol. 7 (1958), pp. 225 to 230). This method consisted in the treatment of human plasma with beta-propiolactone, which could be combined with ultraviolet irradiation. It was also called "cold sterilization" because it was performed at temperatures below 37.degree. C.
The application of the sterilization conditions given by LoGrippo, however, results in high losses of the factor VIII activity in plasma, as shown in the following Table I.degree..
TABLE I ______________________________________ The effect of LoGrippo's sterilization conditions on factor VIII activity in plasma. Factor VIII in % of normal ______________________________________ Fresh citrate plasma 100 After treatment with beta- propiolactone (0.3%) 55 After ultraviolet radiation (2 mW/cm.sup.2 /min) 30 ______________________________________
Only inadequate yields of cryoprecipitate can be obtained from plasma treated with beta-propiolactone and ultraviolet. The combination of beta-propiolactone with ultraviolet treatment of extracts of cryoprecipitate is possible, but under the conditions given by LoGrippo it results in great losses of the Factor VIII activity.
J. T. Sgouris et al., "Stability of Factor I (Fibrinogen) and Factor VIII (Antihemophilic Globulin) to Beta-Propiolactone", Vox Sang., vol. 8 (1963), pp. 105 to 108, W. Doleschel et al., "The Influence of Beta-Propiolactone on the Coagulability of Human Fibrinogen", Pharmacology, vol. 2 (1969), pp. 1 to 8; and H. Brunner et al., "Experimentelle Fibrinogenopathie nach Reaktion von humanem Fibrinogen mit .beta.-Propiolactone: Antithrombinaktivitat, Wirkung auf die Plattchenaggregation und Funktion auf die Fibrinverfestigungsphase", Verhandlungen der Deutschen Gesellschaft fur Innere Medizin, vol. 79 (1973), pp. 1130 to 1134, attempted to use beta-propiolactone for the sterilization of coagulation factors, especially protein fractions containing factor VIII and fibrinogen.
It was found, however, as a result of this work that the method described by LoGrippo for the sterilization of plasma or serum with beta-propiolactone and ultraviolet radiation is not appropriate for the production of fibrinogen or of preparations containing factor VIII, since these proteins either suffer excessive activity losses or the beta-propiolactone concentration given by LoGrippo has to be reduced to such an extent that a sufficient viricidal action is not realized.
In the production of vaccines it is common to combine different inactivation methods for the inactivation of viruses, for the purpose of eliminating "resistant fractions" which might be resistant to one particular method of inactivation, but which are inactivated by a second or third treatment whose inactivation mechanism is different from that of the first treatment.